热疗上调细胞间隙连接通讯对胶质瘤侵袭性的影响及其机制

目的探讨热疗降低胶质瘤侵袭性的作用与细胞间隙连接通讯(gap junctional intercellular communication,GJIC)的关系。方法热疗处理C6胶质瘤细胞后,免疫组织化学法和Western blot法动态检测HSP70和Cx43的表达水平;划痕标记染料示踪技术检测胶质瘤GJIC功能变化;结晶紫染色法检测胶质瘤侵袭性的改变。结果 C6细胞经热疗后,HSP70表达增加,于30 min时含量最多。C6细胞内Cx43的表达水平也在热疗后明显增加,并于热疗后的120 min达高峰,后逐渐减少。热疗后GJIC功能的恢复与C6细胞内Cx43的表达相一致,且GJIC功能越强,胶质瘤侵袭性越低。结论胶质瘤细胞经热疗后HSP70表达增加,增加的HSP70可能是通过其分子伴侣作用提高Cx43的表达水平,进而上调GJIC功能而引起胶质瘤侵袭性的下降。     

缝隙连接蛋白43对人非小细胞肺癌HCC827细胞吉非替尼获得性耐药机制的初步研究

目的探讨缝隙连接蛋白43(connexin 43,Cx43)与非小细胞肺癌(NSCLC)细胞产生吉非替尼(Gefitinib)获得性耐药的关系。方法在Gefitinib敏感细胞株HCC827上,通过逐步递增Gefitinib浓度诱导获得Gefitinib耐药细胞株HCC827 GR;MTT法检测Gefitinib对HCC827/GR细胞的IC_(50);RT-PCR检测Cx43的mRNA水平;Western blot检测Cx43和磷酸化Akt(p-Akt)的蛋白水平;parachute荧光示踪法检测细胞缝隙连接功能(gap junction intercellular communication,GJIC);免疫荧光技术检测Cx43的细胞定位。结果Gefitinib作用于HCC827和HCC827 GR的IC_(50)分别为(0.07±0.019)μmol·L~(-1)、(10.84±0.021)μmol·L~(-1)(P<0.01);HCC827 GR中Cx43的mRNA和蛋白水平较HCC827明显降低(P<0.05),但p-Akt蛋白水平明显升高(P<0.05)。在HCC827 GR细胞上加PI3K的特异性抑制剂LY294002(25μmol·L~(-1),24 h)后,p-Akt蛋白水平明显下降(P<0.01),且Cx43的蛋白水平明显升高(P<0.01)。HCC827及HCC827 GR均未检测到GJIC,用GJIC增强剂RA(retinoic acid)处理(10μmol·L~(-1),24 h)上述细胞,亦未检测到荧光传递,免疫荧光结果显示Cx43表达在细胞胞质。结论胞质中Cx43的下调可能促进NSCLC细胞对Gefitinib的获得性耐药,其机制可能与Cx43非GJIC依赖的PI3K/Akt信号通路激活有关。     

Cx43表达与多发性骨髓瘤细胞耐药的相关性

目的探讨骨髓间充质干细胞(BMSCs)连接蛋白43(Cx43)表达对多发性骨髓瘤(MM)RPMI 8226细胞耐药的影响。方法体外分离、培养BMSCs,采用RT-PCR法检测BMSCs、RPMI 8226细胞Cx43的表达,流式细胞术和多重液相蛋白定量技术分别检测BMSCs与RPMI 8226细胞共培养对RPMI 8226细胞凋亡和细胞因子分泌的影响。结果 BMSCs及RPMI 8226细胞均表达Cx43。BMSCs与RPMI 8226细胞共培养后,上清液中IL-6、IL-10和TGF-β水平增加,硼替佐咪诱导的RPMI 8226细胞凋亡作用减弱;而细胞间隙连接通讯(GJIC)特异性阻断剂18α甘草次酸(18α-GA)能明显逆转上述观察指标的改变过程。结论 BMSCs与RPMI 8226细胞间可形成功能性GJIC,是BMSCs促进MM细胞生存及介导其耐药的重要原因之一。     

复方藤梨根制剂调节PG细胞间隙连接蛋白Cx43基因表达水平的研究

目的探讨复方藤梨根制剂对高转移性人肺癌细胞(PG)的生长及细胞间隙连接蛋白Cx43表达水平的影响。方法采用MTT法体外观察不同浓度复方藤梨根制剂对PG细胞的杀伤作用;应用流式细胞仪检测用药后PG细胞Cx43的表达水平。结果4个浓度的复方藤梨根制剂对PG细胞均有杀伤作用,呈剂量依赖关系。与对照组相比,Cx43表达水平逐渐上升。结论复方藤梨根制剂对PG细胞具有生长抑制作用,其机制可能与上调间隙连接蛋白Cx43表达水平有关。     

复方藤梨根制剂调节PG细胞间隙连接蛋白Cx43基因表达水平的研究

目的探讨复方藤梨根制剂对高转移性人肺癌细胞(PG)的生长及细胞间隙连接蛋白Cx43表达水平的影响。方法采用MTT法体外观察不同浓度复方藤梨根制剂对PG细胞的杀伤作用;应用流式细胞仪检测用药后PG细胞Cx43的表达水平。结果4个浓度的复方藤梨根制剂对PG细胞均有杀伤作用,呈剂量依赖关系。与对照组相比,Cx43表达水平逐渐上升。结论复方藤梨根制剂对PG细胞具有生长抑制作用,其机制可能与上调间隙连接蛋白Cx43表达水平有关。     

玉米赤霉烯酮对细胞间隙连接通讯的影响

目的 了解真菌性雌激素玉米赤霉烯酮(ZEA)对细胞间隙连接通讯(GJIC)的影响。方法 用荧光漂白后恢复(fluorescence redistribution after photobleaching，FRAP)技术，在激光扫描共聚焦显微镜下观察ZEA对HaCaT细胞GJIC功能的影响。结果 ZEA在0．1μmol/L对HaCaT细胞的GJIC没有明显影响，但在I—100μmol/L浓度下都有明显的抑制作用。ZEA不能有效地拮抗TPA引起的GJIC抑制。结论 ZEA在1μmol/L以上就能抑制GJIC功能，提示它在一定条件下可能有促癌作用。     

大黄素对Cx32组成的细胞缝隙连接通讯功能的影响

目的在转染并稳定表达Cx32的Hela细胞上,观察大黄素对Cx32的GJIC以及Cx32蛋白表达水平的影响。方法采用SRB法检测不同浓度大黄素对Hela细胞的毒性;用细胞接种荧光示踪法("parachute"dye-couplinga ssay)观察不同浓度大黄素对GJIC的影响;用western blot法研究大黄素在影响GJIC功能浓度范围内对Cx32蛋白表达的影响。结果大黄素在0~1μmol/L浓度时对Hela细胞无毒性作用;大黄素(24~600nmol/L)预处理4h,能浓度依赖性地增强GJIC及Cx32蛋白表达量。结论大黄素能够增强Cx32的细胞GJIC;此增强作用可能与其增加Cx32蛋白表达水平有关。     

吴茱萸次碱对棕榈酸诱导的脂质损伤肝细胞的影响

目的:研究吴茱萸次碱(Rut)对棕榈酸(PA)诱导的脂质损伤肝细胞(LO2)的细胞活力、活性氧(ROS)以及缝隙连接蛋白32(Cx32)和缝隙连接蛋白26(Cx26)表达水平的影响。方法:加入不同浓度的Rut溶液预处理LO2细胞20 min后,每个孔加入PA(0.2 mmol/L)溶液,共同孵育24 h,分别应用细胞增殖及毒性检测试剂盒(CCK-8)检测细胞活力,二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)荧光探针检测细胞内活性氧的表达、划痕负载实验检测缝隙连接细胞间通讯(GJIC)的功能变化及免疫印迹实验(Western blotting)检测缝隙连接蛋白Cx32和Cx26的表达水平。结果:CCK-8实验结果显示,Rut(0.1μmol/L,0.5μmol/L,1μmol/L)能抑制PA诱导的LO2细胞活力的下降;DCFH-DA荧光探针实验显示PA诱导细胞后ROS的表达增强,Rut预处理细胞后能够减少ROS的表达;划痕负载实验结果显示,PA诱导细胞后能够抑制细胞的GJIC功能,预先给予Rut处理能够改善GJIC的功能障碍;Western blotting显示PA诱导细胞后缝隙连接蛋白Cx32和Cx26的表达水平显著降低,给予Rut预处理后能够恢复Cx32和Cx26的表达。结论:PA能够诱导LO2细胞的脂质损伤,导致细胞活性降低、细胞内活性氧增加以及GJIC的功能障碍;Rut预处理LO2细胞后可减轻PA诱导的细胞损伤,并且恢复细胞的缝隙连接蛋白Cx32和Cx26的表达和GJIC的功能。     

赖氨匹林增强Cx32/Cx26组成的Hela细胞缝隙连接功能

目的 体外观察赖氨匹林对转染并稳定表达连接蛋白32/26 （Cx32/Cx26）的Hela细胞缝隙连接通讯（GJIC）功能和Cx32/Cx26蛋白表达水平的影响.方法 不同浓度（0、1、5、10 mmol·L^-1）赖氨匹林作用于人宫颈癌Hela细胞24 h后,以划痕标记/染料示踪技术（SL/DT）检测赖氨匹林作用Hela细胞48 h后荧光黄在细胞之间传递的距离来评价其对缝隙连接（GJ）功能的影响;Western blot方法检测赖氨匹林对Cx32/Cx26蛋白表达的影响.结果 SL/DT检测显示赖氨匹林具有上调GJ功能的作用;Western blot显示赖氨匹林增加Cx32/Cx26蛋白表达水平.结论 赖氨匹林抑制转染并稳定表达Cx32/Cx26的Hela细胞的增殖、上调GJ功能且这种作用可能与其增加Cx32/Cx26蛋白表达水平有关.     

以siRNA表达载体稳定抑制缝隙连接蛋白43表达的睾丸间质细胞和睾丸支持细胞系的建立

目的构建Cx43-siRNA真核表达载体,获得连接蛋白43(connexin43,Cx43)被长期稳定抑制的睾丸间质细胞(TM3细胞)系和睾丸支持细胞(TM4细胞)系,为研究Cx43及其形成的细胞缝隙连接(gap junction,GJ)在睾丸组织中的作用提供有用模型。方法设计合成3对针对Cx43的短发夹样siRNA的DNA模板序列,定向连接到siRNA真核表达载体pSilencerTM2.1-U6neo上,通过测序鉴定后以脂质体法瞬时转染睾丸支持细胞,以Western blot方法检测Cx43蛋白表达水平,筛选出最有效的干扰序列,再将之分别转染睾丸间质细胞和睾丸支持细胞,G418筛选出能稳定表达siRNA的细胞系,以"Parachute"荧光传递示踪法检测细胞缝隙连接功能。结果 Western blot结果显示,第3对干扰序列对Cx43表达抑制效果最佳,以表达该序列的质粒稳定转染的TM3细胞和TM4细胞上Cx43蛋白表达水平均明显降低;荧光传递示踪法检测表明,两种细胞系的GJ功能均被明显抑制。结论以Cx43-siRNA真核表达载体稳定转染的方法能长期干扰TM3和TM4细胞上Cx43的表达,并抑制由其形成的GJ功能。     

Tumor promoters induce inhibition of gap junctional intercellular communication in mouse epidermal cells by affecting the localization of connexin43 and E-cadherin

The molecular and histological effects of tumor promoters on gap junctional intercellular communication (GJIC) were studied in three mouse epidermal cell types, representing different stages of tumor formation. GJIC was inhibited by most of the studied compounds (l-ethionine, d-limonene, o-anisidine, clofibrate, Aroclor 1260 and 1,1'-(2,2,2-trichloroethylidene)bis(4-chlorobenzene) (DDT)) except NaF and phenobarbital (PB). Whatever their effect on GJIC, most of the studied compounds increased the phosphorylation state of the gap junction protein expressed in these cells, connexin43 (Cx43), as shown by Western analysis. All agents with GJIC inhibiting capacity changed the intensity of the immunofluorescent staining of Cx43 on the membrane of the cells, whereas NaF and PB had no effect on Cx43 immunostaining. No association could be found between the type of change in Cx43 localization (changed membrane and/or cytosolic staining) and Cx43 phosphorylation or GJIC inhibition. Because the cell adhesion molecule E-cadherin also regulates GJIC, the effects of tumor promoters on E-cadherin protein and localization were studied. No quantitative change could be observed in E-cadherin protein content of cells treated with any of the selected agents. However, all agents which decreased GJIC, affected E-cadherin immunostaining of the membrane, while PB and NaF had no effect. These results show that an association exists between inhibition of GJIC and localization of both connexin43 and E-cadherin protein, but not with Cx43 phosphorylation.     

TPA-induced inhibition of gap junctional intercellular communication is not mediated through free radicals

The present investigation was designed to determine whether the inhibition of gap junction-mediated intercellular communication (GJIC) induced by TPA (12-O-tetradecanoylphorbol-13-acetate) in rat liver epithelial (WB-F344) cells in vitro is mediated through free radical production. As assessed by fluorescence redistribution after photobleaching (FRAP) analysis, GJIC was significantly inhibited in cells treated for 1 hr with either 10 ng/ml TPA or 500 microM hydrogen peroxide (H2O2). Addition of 1000 U/ml catalase or 25 microM N',N'-diphenyl-p-phenylenediamine (DPPD) to TPA-treated cells did not alleviate the TPA-induced inhibition of GJIC. However, the concurrent addition of 1000 U/ml catalase to the culture medium prevented the H2O2 inhibition of GJIC. 2'-7'-dichlorofluorescein-mediated fluorescence, a measure of free radical production utilizing the Meridian ACAS 470 interactive laser cytometer, was not significantly increased in WB-F344 cells treated with 10 and 100 ng/ml TPA when compared to control cells. However, polymorphonuclear leukocytes (PMNs) treated for 10 min with 100 ng/ml TPA showed a substantial oxidative burst, as did WB-F344 cells treated for 1 hr with 500 microM H2O2. The concurrent addition of 1000 U/ml catalase to the culture medium attenuated H2O2-mediated free radical production in both PMNs and WB-F344 cells. Data from this study do not support a role for free radicals in the TPA-induced inhibition of GJIC in WB-F344 cells.     

The 2,2',4,4',5,5'-Hexachlorobiphenyl-Enhanced Degradation of Connexin 43 Involves Both Proteasomal and Lysosomal Activities

One of the toxic effects of non-dioxin–like polychlorinatedbiphenyls (NDL-PCBs) is the acute inhibition of gap junctionalintercellular communication (GJIC), an event possibly associatedwith tumor promotion. The model NDL-PCB-2,2',4,4',5,5'-hexachlorobiphenyl(PCB 153)—induces a sustained GJIC inhibition in rat liverepithelial WB-F344 cells. As this effect might be related toderegulation of connexin 43 (Cx43) synthesis, trafficking, ordegradation, we investigated the impact of PCB 153 on theseevents. Although PCB 153 had no effect on Cx43 mRNA levels,it induced a gradual loss of Cx43 protein and significantlydecreased the amount of gap junction plaques in plasma membrane.PCB 153 contributed to extracellular signal–regulatedkinases 1 and 2 (ERK1/2)–dependent accumulation of hyperphosphorylatedCx43-P3 form, thus indicating that ERK1/2 activation by PCB153 might contribute to its effects on Cx43 internalizationor degradation. Inhibition of either proteasomes or lysosomeswith their specific inhibitors largely restored total Cx43 proteinlevels, thus suggesting that both proteasomes and lysosomesmay participate in the PCB 153–enhanced Cx43 internalizationand degradation. However, neither the proteasomal nor the lysosomalinhibitors restored normal GJIC or number/size of gap junctionplaques. Finally, PCB 153 also interfered with restoration ofgap junction plaques following the inhibition of Cx43 transportto plasma membrane. Taken together, multiple modes of actionseem to contribute to downregulation of Cx43 in PCB 153–treatedrat liver epithelial cells. The enhanced degradation of Cx43,together with persistent inhibition of GJIC, might contributeto tumor-promoting effects of NDL-PCBs.     

(-)-Epicatechin effects in rat liver epithelial cells: stimulation of gap junctional communication and counteraction of its loss due to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate

Gap junctional intercellular communication (GJIC) is a direct signaling pathway for neighboring cells. Disturbances in GJIC are suggested to play a role in carcinogenesis and may be involved in cardiac arrhythmia. Tumor promoters like 12-O-tetradecanoylphorbol-13-acetate (TPA) are capable of inhibiting GJIC, whereas GJIC is stimulated by several micronutrients like genistein, retinoids or carotenoids. (-)-Epicatechin (4-40 microM), a major flavonoid in cocoa and green tea, exhibited stimulatory effects on GJIC in WB-F344 rat liver epithelial cells after 24-72hr of incubation; no change was observed after 90 min. However, treatment of cells for 90 min with TPA (5 or 10nM) led to complete loss of GJIC, whereas 40% loss was found with 1nM. These inhibitory effects of TPA were largely suppressed when (-)-epicatechin or genistein (40 microM) were present during the incubation. In communicating WB-F344 cells, most of the major gap junction protein connexin43 (Cx43) was located in the plasma membrane. When the cells were exposed to TPA, considerably less protein was found in the membrane. Such a delocalization of Cx43 proteins was not observed when TPA was coincubated with the flavonoids, (-)-epicatechin or genistein. It is concluded that TPA affects Cx43 trafficking between cellular compartments, and that this effect is counteracted by (-)-epicatechin or genistein.     

Chemopreventive effect of dimethyl dicarboxylate biphenyl on malignant transformation of WB-F344 rat liver epithelial cells

AIM: To study the potential chemopreventive effect of dimethyl dicarboxylate biphenyl (DDB), an anti-hepatitis drug, on hepatocarcinogenesis in vitro. METHODS: The anti-carcinogenesis effect of DDB was assessed on a two-stage chemical oncogenesis model induced by 3-methylcholanthrene and 12-O-tetradecanoyl phorbol 13-acetate (TPA) with WB-F344 rat liver epithelial cells (WB-F344 cells) in vitro. A soft-agar colony formation assay was used to determine the tumorigenic potential of the transformed WB-F344 cells. The gap junctional intercellular communication (GJIC) was detected using the scrape loading/dye transfer technique. RESULTS: DDB at 1 micromol/L, 2 micromol/L, and 4 micromol/L significantly prevented the malignant transformation of WB-F344 cells induced by 3-methylcholanthrene and TPA. The average number of transformed foci decreased dramatically by 10.0%, 37.2%, and 47.4%, respectively. In soft agar, a remarkable decrease in colony numbers was observed in transformed cells treated with 2 micromol/L and 4 micromol/L DDB. DDB at 1 micromol/L, 2 micromol/L, and 4 micromol/L inhibited the downregulation of GJIC induced by TPA in a dose-dependent manner. The GJIC recovered to 25.6%, 34.6%, and 44.9%, respectively, of the control WB-F344 cells by DDB. CONCLUSION: DDB has a potential chemopreventive effect on hepato-carcinogenesis induced by carcinogens in vitro.     

Effects of cadmium on gap junctional intercellular communication in WB-F344 rat liver epithelial cells

Cadmium has been associated with a number of tumors but its role in tumor promotion has not been elucidated clearly or the results obtained from various studies have been conflicting. This study was designed to investigate the effects of cadmium on the gap junctional intercellular communication (GJIC), number of gap junctions per cell, and cell proliferation in WB-F344 rat liver epithelial cells from the viewpoint of tumor promotion. GJIC was monitored by counting the cells stained with Lucifer yellow CH dye, using the scrape-loading and dye-transfer method. The numbers of gap junctions per cell were visually quantitated after an indirect immunostaining for gap junction protein using an antibody to connexin 43. Cell proliferation was assayed by direct counting of the living cells using the trypan blue dye exclusion method. In the time course study, cells treated with 200 microM CdCl2 showed rapid and nearly complete inhibition of GJIC (approximately 14% of the control) and a decrease in the number of gap junctions per cell (approximately 21% of the control) at 30 min, and the decrease continued up to 4 h without any changes in the cell viability. Treatment with CdCl2 (7.4-200 microM) for 4 h resulted in the decrease of GJIC and gap junction numbers per cell in a dose-response pattern without changes in the cell viability. In the long-term (14 days) exposure studies at doses of 0.01-7.4 microM CdCl2, an increase in cell proliferation was observed at low doses of 0.03-2.5 microM CdCl2, with GJIC also decreasing. These data demonstrate that cadmium inhibits GJIC, reduces the number of gap junctions per cell, and induces cell proliferation while decreasing the function of the gap junction.     

Influence of tris(4-chlorophenyl)methanol (TCPM) on gap junction-mediated intercellular communication of cultured bovine granulosa cells.

Tris(4-chlorophenyl)methanol (TCPM) is a by-product in the manufacture of technical grade DDT, which is known to alter properties and functions of the female reproductive system. We investigated whether in vitro TCPM has an influence on the function of gap junction-mediated intercellular communication (GJIC) and gap junction protein expression of connexin 43 (Cx43) in cultured bovine granulosa cells. GJIC was assessed by fluorescent dye microinjection (dye-coupling). After a 1-h exposure to TCPM at a concentration of 32 microM, a significant (P<0.05) reduction in dye coupling occurred. The same result was obtained with o,p'-DDT. At a concentration of 32 microM both pesticides were cytotoxic as indicated by significant (P<0.05) increased propidium iodide staining of the cell nuclei. Little or no effect on the stainable pattern of connexons occurred after 1 h incubation time, while after 3 h treatment from 16 to 64 microM TCPM, a significant inhibition in the immunostaining resulted and the concentrations of 32 and 64 microM TCPM were cytotoxic for the granulosa cells. The freeze-fracture electron microscopy resulted in small differences in the morphology of gap junction plaques of cell cultures treated for 3 h with 8 or 16 microM TCPM in comparison to untreated cells. After treatment with 32 microM TCPM, gap junction plaques were very rarely detected and the lateral intramembraneous particles (IMP) distribution of many plasma membranes was strongly altered. Estimation of the cellular parameters may lead to an enhanced understanding of the mechanism of chemically induced toxicity by TCPM, that causes a general toxic effect on granulosa cells. We can conclude that TCPM is a toxic risk in the same manner as DDT.     

Toxic effects of methylated benz[a]anthracenes in liver cells

Monomethylated benz[ a]anthracenes (MeBaAs) are an important group of methylated derivatives of polycyclic aromatic hydrocarbons (PAHs). Although the methyl substitution reportedly affects their mutagenicity and tumor-initiating activity, little is known about the impact of methylation on the effects associated with activation of the aryl hydrocarbon receptor (AhR)-dependent gene expression and/or toxic events associated with tumor promotion. In the present study, we studied the effects of a series of MeBaAs on the above-mentioned end points in rat liver cell lines and compared them with the effects of benz[ a]anthracene (BaA) and the potent carcinogen 7,12-dimethylbenz[ a]anthracene (DMBA). Methyl substitution enhanced the AhR-mediated activity of BaA derivatives determined in a reporter gene assay, as the induction equivalency factors (IEFs) of all MeBaAs were higher than that of BaA. IEFs of 6-MeBaA and 9-MeBaA, two of the most potent MeBaAs, were more than two orders of magnitude higher than the IEF of BaA. Correspondingly, all MeBaAs induced higher levels of cytochrome P450 1A1 mRNA. Both BaA and MeBaAs had similar effects on the expression of cytochrome P450 1B1 or aldo-keto reductase 1C9 in rat liver epithelial WB-F344 cells. In contrast to genotoxic DMBA, MeBaAs induced low DNA adduct formation. Only 10-MeBaA induced apoptosis and accumulation of phosphorylated p53, which could be associated with the induction of oxidative stress, similar to DMBA. With the exception of 10-MeBaA, all MeBaAs induced cell proliferation in contact-inhibited WB-F344 cells, which corresponded with their ability to activate AhR. 1-, 2-, 8-, 10-, 11-, and 12-MeBaA inhibited gap junctional intercellular communication (GJIC) in WB-F344 cells. This mode of action, like disruption of cell proliferation control, might contribute to tumor promotion. Taken together, these data showed that the methyl substitution significantly influences those effects of MeBaAs associated with AhR activation or GJIC inhibition.